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Biomedical subjects

E F Knol

Publications and source records attributed to E F Knol.

At least 73 records · Page 4Linked to original sources
PubMed1996

Transmigration of human neutrophils across airway epithelial cell monolayers is preferentially in the physiologic basolateral-to-apical direction.

To study the mechanisms involved in the movement of neutrophils from the blood stream into the lung airways, we investigated human neutrophil transmigration across a monolayer of human airway epithelial cells, both in the apical-to-basolateral direction and in the more physiologic basolateral-to-apical direction. Migration of human neutrophils across monolayers of human airway epithelial H292 cell-line cells and primary bronchial epithelial cells occured most efficiently in the basolateral-to-apical direction, both after the addition of chemoattractants to resting epithelial cells and across interleukin-1beta (IL-1beta)-stimulated epithelial cells. Blocking studies with monoclonal antibodies revealed that the migration of neutrophils was mediated by the CR3 adhesion molecule (CD11b/CD18) on the neutrophils. IL-1beta-treated epithelial cells caused neutrophil movement via the secretion of chemoattractants. The most potent chemoattractant released by the epithelial cells was found to be IL-8, because the IL-1beta-induced migration was inhibited for 75 +/- 10% by the addition of an antibody against IL-8. After apical stimulation of the epithelial cells with an optimal concentration of IL-1beta, 27 +/- 4 ng/ml IL-8 was found in the supernatant at the apical side of epithelial cells. Platelet-activating factor (PAF) synthesis by the epithelial cells did not play a role in neutrophil transmigration, as was demonstrated by the lack of inhibition of this process after addition of the PAF-receptor antagonist WEB 2086. We conclude that the movement of neutrophils across airway epithelial cell monolayers occurs preferentially in the physiologic basolateral-to-apical direction, indicating that the polarity of epithelial cells is important for neutrophil transmigration.

CD18 Antigens↗
PubMed1996

The role of basophils in allergic disease.

During allergic disease, leucocytes infiltrate the affected tissues and release their mediators and cytokines. In this way, the local inflammatory process is induced and maintained. Basophilic granulocytes have been demonstrated in lung and sputum of allergic asthmatics, in nasal mucosa and secretion of allergic rhinitis patients, and in skin lesions of atopic dermatitis patients. The number of basophils correlates with the severity of the disease. Analysis of mediator profiles and cellular contents of lavages of nose, skin and lung during allergic late-phase reactions (LPR) have demonstrated histamine, but not tryptase or prostaglandin D2. The histamine-containing cells have been characterized as basophilic granulocytes. This indicates that infiltrating basophils but not mast cells are activated and release their inflammatory contents in the LPR. We are interested in the cellular mechanisms that determine the degranulation of basophils during LPR. Basophil activators, such as allergens and activated complement, are not present at these sites. However, cytokines that prime basophils but do not induce degranulation, such as interleukin-5 (IL-5) and granulocyte/macrophage colony-stimulating factor (GM-CSF), have been detected at sites of LPR. We have now observed that after emptying intracellular Ca2+ stores by means of the Ca2+ adenosine triphosphatase (ATPase) inhibitor, thapsigargin, basophils become extremely sensitive to stimuli that do not affect the Ca2+ stores themselves but that induce degranulation, such as the phorbolester, phorbol myristate acetate (PMA). The most interesting finding was that although both thapsigargin and IL-3, IL-5 or GM-CSF do not induce basophil degranulation by themselves, a 2 min preincubation of basophils with thapsigargin followed by addition of one of these cytokines resulted in extensive histamine release: IL-3 induced 71 +/- 7% histamine release (conc1/2max 6 pM), IL-5 induced 43 +/- 8% histamine release (conc1/2max 41 pM) and GM-CSF induced 57 +/- 10% histamine release (conc1/2max 140 pM). Interestingly, the effect of thapsigargin could be mimicked by platelet-activating factor (PAF) (range 10(-9) to 10(-6) M), although to a lesser extent. Our results indicate that basophil degranulation in tissues during late-phase reactions might be caused by a combination of mediators or cytokines depleting Ca2+ stores, as platelet-activating factor or thapsigargin do, concurrent with activation by interleukin-3, interleukin-5 or granulocyte/macrophage colony-stimulating factor. The response of the basophils towards these cytokines might also be influenced by cell adhesion events, such as binding of basophils via integrins. This is the subject of further study.

Asthma↗
PubMed1996

Mechanisms regulating eosinophil extravasation in asthma.

Infiltration of eosinophils into tissue allergic inflammation is mediated by a combination of processes. Eosinophil L-selection and very late activation antigen (VLA-4) can selectively regulate eosinophil adhesion to the endothelium. Activating cytokines, such as interleukin (IL)-5, regulated upon activation in normal T-cells (RANTES) and monocyte chemotactic peptide (MCP)-3 specifically act on eosinophils. Moreover, eosinophils from allergic individuals can be primed for increased adhesion and movement by chemokines released at sites of allergic inflammation. Together, these processes induce a specific infiltration of eosinophils.

Animals↗
PubMed1996

The effect of salmeterol and nimesulide on chemotaxis and synthesis of PAF and LTC4 by human eosinophils.

Salmeterol is a long-acting beta 2-adrenoceptor agonist, with several antiasthma properties. Nimesulide is a nonsteroidal anti-inflammatory drug, supposed to act by inhibition of phosphodiesterase type IV. This might indicate that the effects of both drugs are mediated by an increase in cyclic adenosine monophosphate (cAMP). For salmeterol, it has been shown that it inhibits the influx of eosinophils into the lungs of guinea-pigs after platelet-activating factor (PAF) challenge, suggesting an effect of cAMP on eosinophil migration. For neutrophils, it has been shown that PAF synthesis is inhibited by cAMP. In the present study, we have, therefore, measured the effect of salmeterol and nimesulide on two important human eosinophil functions: chemotaxis; and synthesis of PAF and leukotriene C4 (LTC4). Both drugs were found to be inhibitors of the chemotactic responses of human eosinophils. However, at comparable concentrations, only nimesulide was able to inhibit the synthesis of PAF and LTC4 in activated eosinophils. These results indicate that although the effects of both drugs are thought to be mediated by an increase in cyclic adenosine monophosphate, they have differential effects on eosinophil chemotaxis and synthesis of lipid mediators.

Adrenergic beta-Agonists↗
PubMed1995

Eosinophils isolated with two different methods show different characteristics of activation.

Eosinophils can be isolated from a mixed suspension of granulocytes by different procedures. We compared functional responses of human eosinophils purified according to two different principles: (1) an fMLP-induced difference in specific gravity between eosinophils and neutrophils and (2) selective removal of neutrophils by means of immunomagnetic beads coated with CD16 mAb. The results showed that eosinophils isolated with the CD16 beads method have a higher capacity to synthesize platelet activating factor (PAF) after stimulation with serum-treated zymosan (STZ) than eosinophils purified with the fMLP method. Binding of STZ and subsequent activation of the respiratory burst were also increased in CD16-isolated eosinophils. Furthermore, eosinophils isolated with the CD16 beads showed stronger chemotactic responses towards C5a and PAF. The difference in activity of these eosinophil preparations might be explained by a loss of the more active cells during the isolation with the fMLP method: only 30-60% of the eosinophils were recovered with this method, in contrast to a recovery of more than 95% with the CD16 beads method. Indeed, this 'lost' population of eosinophils, subsequently purified with CD16-coated beads, had a higher respiratory burst activity. The alternative explanation, i.e., an enhancement of eosinophil function by the beads method, appeared not to be valid, because repurification of fMLP-isolated eosinophils in the presence of fresh neutrophils and CD16-coated beads did not change the reactivity of the eosinophils. We conclude that the fMLP method leads to selective purification of eosinophils with a resting (or 'unprimed') phenotype.

Antigens, CD↗
PubMed1994

Comparison of human eosinophil and neutrophil adhesion to endothelial cells under nonstatic conditions. Role of L-selectin.

To simulate adhesion that occurs under conditions of flow, we investigated the attachment of eosinophils to endothelium under rotational conditions. Tissue-culture plates containing monolayers of HUVEC were placed on a horizontal rotator (80 revolutions per minute (rpm)), and equal numbers of purified human eosinophils or neutrophils were added to separate wells at 4 degrees C. Binding of eosinophils and neutrophils to unstimulated endothelial cells was 15 +/- 3 and 31 +/- 11 cells/four high power fields (HPF), respectively. After preincubation of HUVEC with IL-1 beta (1 ng/ml, 4 h, 37 degrees C), adhesion increased to 56 +/- 4 and 290 +/- 26 cells/four HPF, respectively (p < 0.0002 for both, n = 8-14). Eosinophils with reduced levels of L-selectin (blood eosinophils activated in vitro or eosinophils obtained from bronchoalveolar lavage (BAL) performed after segmental lung allergen challenge of allergic subjects) demonstrated reduced binding under rotating conditions. Several L-selectin Abs inhibited adhesion of eosinophils and neutrophils (e.g., LAM1-3: 43 +/- 14% vs 63 +/- 3% inhibition; LAM1-6: 73 +/- 5% vs 36 +/- 6% inhibition, respectively, n > or = 6). Interestingly, one additional L-selectin Ab, LAM1-11, inhibited eosinophil but not neutrophil adhesion (51 +/- 2% vs 1 +/- 7% inhibition, respectively, n > or = 5). We conclude that eosinophils, like neutrophils, use L-selectin to bind to activated endothelial cells under conditions of flow, although mAb LAM1-11 can selectively inhibit eosinophil attachment to stimulated endothelial cells in vitro, suggesting different functional epitopes on L-selectin among eosinophils and neutrophils.

Cell Adhesion↗
PubMed1994

Variability of IgE-dependent histamine-releasing activity in supernatants of human mononuclear cells.

Histamine-releasing factors (HRF) that release mediators from human basophils by interacting with IgE have been identified from different cell sources, including lymphocytes, monocytes, thrombocytes and endothelial cells. These factors are studied in view of their potential importance as a stimulus in chronic inflammation. In this report we investigated the qualitative variability of the histamine-releasing activity in the supernatants of activated mononuclear cells. Purified human mononuclear cells of 8 donors were activated with streptokinase/streptodornase (SK/SD) and the supernatants (HRF-MN) were tested for histamine-releasing activity (HRA) in both allergic (RAST positive for inhalant allergens) and nonallergic individuals. Four of the eight HRF-MN supernatants were discriminating, i.e. showing no histamine-release response with nonallergic individuals, whereas four supernatants were not. Two of the HRF-MN supernatants that exhibited discriminating properties were studied in more detail. The response to HRF-MN was tested (1) in a direct bioassay on basophils of allergic (RAST positive for inhalant allergens) and nonallergic individuals and (2) in an indirect bioassay with 70% pure basophils of RAST-negative donors after passive sensitization with sera of allergic donors. An association was found between the response to HRF-MN and the RAST for inhalant allergens: none (0/12) of the RAST-negative but 15/22 of the RAST-positive individuals were HRF-MN responders. The IgE dependency of HRF-MN was shown e.g. by inhibition of passive sensitization by preincubating a responder serum with monoclonal antibody (moAb) anti-IgE MH25-1. Our results are in contrast with findings of other investigators who use pooled supernatants and demonstrated HRF-MN responsiveness with both allergic and nonallergic donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Basophils↗
PubMed1993

Stimulation of human basophils results in homotypic aggregation. A response independent of degranulation.

For a better insight into the mechanisms determining the recruitment of human basophilic granulocytes from the circulation to sites of allergic reactions, we studied the homotypic aggregation of these cells. The aggregation was studied with > 95% pure basophil suspensions obtained from peripheral blood in a double-color flow cytometric analysis. Homotypic aggregation was induced by treatment of the basophils with anti-IgE, house dust mite allergen, the chemoattractant FMLP, PMA, or IL-3. The aggregation by anti-IgE was, in part, mediated by interactions with Fc gamma R-II as indicated by 43 +/- 15% inhibition after pretreatment with CD32 antibodies. The aggregation was mediated by beta 2-integrins, as was shown by inhibition of the response by CD18 antibodies. The aggregation induced by anti-IgE, allergen, and PMA displayed comparable kinetics (t1/2 max, 3 to 4 min), in contrast to the degranulation of basophils. FMLP induced the most rapid response (t1/2max, 1.6 min). Inhibition of protein kinase C by staurosporine resulted in a strong (> 90%) inhibition of the PMA-induced aggregation, whereas the FMLP-induced aggregation was more than doubled (from 11.7 +/- 1.9 to 24.4 +/- 1.9%). Staurosporine did not affect the extent of the anti-IgE-induced aggregation, but it induced a retardation of congruent to 10 min. In most experiments, no clear correlation was found between degranulation and aggregation of human basophils. Most strikingly, IL-3 did not induce degranulation but did induce aggregation. Thus, the homotypic aggregation response of human basophils is induced by intracellular signals not necessarily leading to degranulation. This might be important in the physiologic appearance of basophils at sites of allergic late-phase responses or inflammation.

Alkaloids↗
PubMed1992

An immunosuppressive retrovirus-derived hexapeptide interferes with intracellular signaling in monocytes and granulocytes through N-formylpeptide receptors.

We have previously found that the retroviral p15E-derived hexapeptide LDLLFL is a potent inhibitor of the FMLP-induced polarization response that is an early event in chemotaxis of monocytes and granulocytes. We investigated the mechanism of action of LDLLFL. LDLLFL inhibited the changes in [Ca2+]i in response to FMLP, but not to C5a or leukotriene B4. The reverse peptide LFLLDL was not inhibitory. In the presence of LDLLFL, the FMLP dose-response curve shifted to higher concentrations, indicating that LDLLFL interfered with binding of FMLP to its receptor. Indeed, binding of [3H]FMLP to neutrophilic granulocytes was inhibited in the presence of LDLLFL. Furthermore, immunosuppressive LDLLFL homologs also inhibited binding of FMLP to granulocytes, whereas noninhibitory LDLLFL homologs did not. Our results suggest that retroviral p15E and p15E-like factors, which can be found in serum of patients with cancer or chronic upper airway infections, may interfere with the interaction of N-formylpeptides derived from (opportunistic) bacteria, with monocytes and granulocytes. This receptor interference may impair monocyte and granulocyte reactivity toward these agents.

Amino Acid Sequence↗
PubMed1992

An improved method for the purification of basophilic granulocytes from human blood.

Studies on human basophils are hampered by the low number of basophils in peripheral blood. Here we describe the purification of human basophilic granulocytes with immunomagnetic beads to improve an already established method of purification. A 70% pure basophil suspension was incubated with monoclonal antibodies (CD2, CD14, CD16 and CD19) recognizing the contaminating cells. After incubation with magnetic beads coated with goat anti-mouse IgG, the bead-cell rosettes were removed by a magnet. In this way, the basophil purity increased to 94%. The loss of basophils during the bead procedure was about 20%. The amount of histamine per basophil and the spontaneous release of histamine during subsequent incubation of the cells was not altered by the purification procedure. The kinetics and the dose response of histamine release after the addition of anti-IgE or FMLP was also unchanged. Binding of CD63 was not altered, indicating that the purification procedure did not result in activation of the basophils. This improved method for the purification of human basophils should permit the measurement of non-basophil-specific parameters, such as changes in intracellular free Ca2+, without the problem of interference from contaminating cells.

Antibodies, Monoclonal↗
PubMed1992

Intracellular events in anti-IgE nonreleasing human basophils.

Histamine release (HR) after activation of human basophils with anti-IgE demonstrates a great variability among different donors. To elucidate the biochemical basis of this phenomenon, we studied the activation of purified basophils from donors that barely demonstrated HR (less than 7%) after stimulation by anti-IgE, although IgE was present on these cells and formyl-methionyl-leucyl-phenylalanine (fMLP)-induced HR was normal. Basophils from anti-IgE "nonreleasers" demonstrated, in contrast to cells from "releasers," hardly any changes in cytosolic-free Ca++ concentration after addition of anti-IgE. However, anti-IgE treatment of basophils from these anti-IgE nonreleasers resulted in at least two intracellular changes. First, a profound inhibition (54.2% +/- 4.2%) of a subsequent fMLP-induced HR was observed. This inhibition caused by anti-IgE was also observed in basophils from anti-IgE releasers treated with wortmannin, an inhibitor of the anti-IgE-induced HR. The time course of the inhibition induced by wortmannin plus anti-IgE on the fMLP-induced HR (half-life maximum, 4 minutes; time of maximum concentration, 10 minutes) was comparable to the time course of the anti-IgE-induced HR. Second, anti-IgE did induce homotypic aggregation in these anti-IgE-nonreleasing basophils, like in anti-IgE-releasing basophils. These studies indicate that basophils not responding to anti-IgE with HR or cytosolic-free Ca++ concentration changes do generate intracellular signals that inhibit the subsequent response to a heterologous stimulus and induce homotypic aggregation.

Antibodies, Anti-Idiotypic↗
PubMed1991

Differential activation of human basophils by anti-IgE and formyl-methionyl-leucyl-phenylalanine. Indications for protein kinase C-dependent and -independent activation pathways.

Upon activation, basophilic granulocytes release inflammatory mediators such as histamine. We studied histamine release of human basophils (64.1 +/- 9.6% pure) after cross-linking of membrane-bound IgE via anti-IgE, or after binding of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). A variability in the extent of histamine release upon stimulation by either anti-IgE or fMLP was found between donors. Kinetic studies revealed that the histamine release induced by anti-IgE (t1/2 greater than 240 s) was more than 20-fold slower than the almost instantaneous release upon stimulation with fMLP (t1/2 less than 10 s). Differences in the cell activation pathways triggered by these stimuli were further analyzed with staurosporine, an inhibitor of protein kinase C (PKC) and with wortmannin, an inhibitor of a PKC-independent pathway. Inhibition of PKC resulted in a partial inhibition of the anti-IgE-induced release, whereas the fMLP-induced release was slightly potentiated. The anti-IgE-induced release was completely inhibited in the presence of wortmannin. This inhibitor too, had no effect on the fMLP-induced release. We conclude that major differences exist in the signal-response coupling between the anti-IgE and fMLP-induced histamine release in human basophils. The so-called releasability of human basophils may be due to the availability of different cell activation pathways.

Alkaloids↗
PubMed1991

Monitoring human basophil activation via CD63 monoclonal antibody 435.

On activation of human basophilic granulocytes with anti-IgE or with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, the expression of the CD63 antigen on the cell surface, detected by monoclonal antibody (MAb) 435, increased up to 100-fold. The kinetics of CD63 up regulation and histamine release were identical, and a strong correlation was found between percentage of MAb 435-binding basophils and extent of histamine release. Immunoelectronmicroscopy demonstrated that the epitope for MAb 435 in resting basophils is located on the basophilic granule membrane. After basophil activation, MAb 435 bound to the exterior of the plasma membrane. Experiments with various doses of anti-IgE demonstrated that the binding of MAb 435 to basophilic granulocytes follows an all-or-nothing-like response per cell. Basophils either do not bind the MAb at all, or they bind a maximal amount of the MAb. We also measured the up regulation of the CD11/CD18 leukocyte adhesion complex. Here, too, we noted an increase in cell-surface exposure of all subunits after activation. This increase was not as strong as increase found with MAb 435. Thus, MAb 435 is an interesting new tool for investigating the activation of human basophils, in addition to the measurement of mediator release. This MAb may be useful for the detection of basophil activation in vivo.

Antibodies, Monoclonal↗
PubMed1990

Differential mechanisms in the stimulus-secretion coupling in human basophils: evidence for a protein-kinase-C-dependent and a protein-kinase-C-independent route.

Upon activation, basophilic granulocytes release inflammatory mediators, such as histamine. We studied histamine release (HR) of purified (64 +/- 10%) human basophils after cross-linking of membrane-bound IgE via anti-IgE or after binding of the chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). A variability in the extent of histamine release upon stimulation by either anti-IgE or FMLP was found between donors. Non-responders for FMLP showed high histamine release for anti-IgE, and vice versa. Inhibition of protein kinase C (PKC) by staurosporine (STSP) resulted in partial inhibition of the anti-IgE-induced HR, whereas inhibition of a PKC-independent pathway by wortmannin (WTM) totally blocked the anti-IgE induced histamine release. The HR induced by FMLP was not affected by either of these inhibitors. We conclude that major differences exist in the signal-response coupling between the anti-IgE and FMLP-induced HR in human basophils. The so-called releasability of human basophils may be due to the availability of different cell activation pathways.

Alkaloids↗
PubMed1989

Accessory cells with a morphology and marker pattern of dendritic cells can be obtained from elutriator-purified blood monocyte fractions. An enhancing effect of metrizamide in this differentiation.

Human monocytic cell fractions obtained by counterflow elutriation centrifugation (with regard to specific monocyte/macrophage characteristics: 82-88% were positive for nonspecific esterase; 86-94% for CD14) were cultured (overnight, 37 degrees C) under nonadhering conditions (polypropylene tubes). Thirty to 40% of the cells were found to differentiate into large, monocytoid cells with a dendritic morphology. These cells expressed a marker of active dendritic cells RFD1 in 76-89% and were also positive for class II MHC antigens as identified by OKIa (95-97%). An exposure of the blood monocytic cells to metrizamide (30 min, 14.5%) prior to the overnight culture enhanced this differentiation, and 47-58% of cells with a dendritic morphology were found (of which 80-87% RFD1 positive, and 95-97% Class II MHC positive). The cultured cell populations containing the cells with the morphology and marker pattern typifying dendritic cells, appeared functionally more active as accessory populations when compared to freshly isolated blood monocytic cell fractions; the cultured cells had an enhanced stimulator capability in MLR, and cluster formation with lymphocytes was more prominent. At the same time, the cultured cell population showed a decreased bactericidal activity when compared to the freshly isolated monocytic populations, and in addition, all the cultured monocytoid cells had lost non-specific-esterase activity, while only approximately 10% of cells were still positive for the CD14 marker. When U937 cells were exposed to metrizamide (14.5% concentration, 30 min) and cultured under nonadhering conditions for 36 hours, similar changes were observed (30-45% became dendritic, 20% RFD1 positive).(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-Presenting Cells↗